UNIVERSITY   OF   CALIFORNIA 

COLLEGE   OF   AGRICULTURE 

AGRICULTURAL   EXPERIMENT   STATION 

BERKELEY,    CALIFORNIA 

CIRCULAR  310 

November,  1927 

THE  OPERATION  OF  THE  BACTERIOLOGICAL 
LABORATORY  FOR  DAIRY  PLANTS 

C.  S.  MUDGEi 


A  typical  bacteriological  laboratory  for  a  milk  plant. 


INTRODUCTION 

Food  stuffs  generally  are  said  to  be  perishable.  By  this  we  mean 
that  sooner  or  later  the  material  is  rendered  unfit  or  perhaps  undesir- 
able for  human  consumption.  It  was  Pasteur  who  found  that  the 
1  perishability '  of  foods  was  due,  not  to  a  chemical  change,  but  to  the 
action  of  minute  living  bodies  known  as  bacteria.  Koch  later  devised 
a  method  whereby  these  bacteria  might  be  counted,  a  method  that  is 
used  today  with  certain  changes. 


1  Associate  Professor  of  Dairy  Industry  and  Associate  Bacteriologist  in  the 
Experiment  Station. 


2  UNIVERSITY    OF    CALIFORNIA EXPERIMENT    STATION 

Milk  is  one  of  the  foods  that  is  considered  highly  perishable,  since 
bacteria  grow  rapidly  in  it.  The  same  constituents  that  make  milk 
"the  perfect  food"  for  man  also  furnish  the  bacteria  with  an 
abundant  supply  of  the  most  easily  assimilated  food.  Roughly  the 
number  of  bacteria  in  milk  is  a- measure  of  its  quality  and  indicates 
the  methods  used  in  its  production,  so  bacteriologists  early  began  to 
study  milk  from  the  standpoint  of  the  bacteria  present.  Here  and 
there  various  workers  began  to  tell  of  the  numbers  and  kinds  of 
bacteria  they  found.  Unfortunately  each  man  in  his  own  laboratory 
employed  methods  which  were  largely  his  own.  He  used  the  solid- 
gelatin  medium  that  Koch  had  told  about,  but  the  food  material 
might  be  any  soluble  meat  or  vegetable  extract  that  suited  his  fancy. 
With  such  a  variety  of  methods  the  results  obtained  by  different 
bacteriologists  were  not  comparable. 

In  1873  a  group  of  public-health  officials  and  other  prominent 
sanitarians  formed  the  American  Public  Health  Association  to  facili- 
tate the  enlightenment  of  the  general  public  on  matters  of  public 
health  and  to  act  as  a  mentor  for  workers  in  this  field.  Acting  in 
this  capacity  a  committee  in  1895  devised  a  standard  method  for  the 
bacteriological  examination  of  water.  Every  step  in  the  procedure 
from  sampling  to  the  final  report  was  carefully  described.  This 
proved  so  successful  that  those  members  of  the  association  interested 
more  particularly  in  milk,  appointed  a  committee  to  formulate 
standard  methods  especially  adapted  to  milk  analysis.  The  findings 
of  this  committee  have  been  published,  and  it  is  earnestly  suggested 
that  a  cop}^  of  the  pamphlet2  be  obtained  before  an  attempt  is  made 
to  do  bacteriological  work  on  milk. 

The  Standard  Methods  of  Milk  Analysis  of  the  American  Public 
Health  Association,  then,  is  the  result  of  diligent  work  of  these  com- 
mittees. The  methods  are  recognized  as  being,  in  theory  at  least,  just 
what  the  name  indicates — standard.  It  should  be  the  aim  of  all  those 
who  undertake  the  enumeration  of  the  bacteria  in  milk  to  adhere 
to  these  methods  exactly.  Some  workers  believe  that  the  standard 
methods  err.  They  are  prone  to  attribute  the  least  variation  in  the 
results  to  the  method  itself,  forgetting  the  possible  human  factor 
which  enters  into  all  but  the  most  exact  methods.  The  methods  used 
in  enumerating  bacteria  in  milk  are  not  exact ;  they  are  not  intended 
to  be.  Newer  knowledge  of  the  science  of  bacteriology  of  course 
makes  the  method  more  and  more  dependable  and  from  time  to  time 


2  American  Public  Health  Association.  Standard  methods  of  milk  analysis, 
5th  ed.,  40  pp.  American  Public  Health  Association,  370  Seventh  ave.,  New  York 
City.     1926. 


CIRC.   310]  BACTERIOLOGICAL    LABORATORY    FOR   DAIRY    PLANTS  3 

changes  are  made  in  the  methods  which  in  the  opinion  of  the  sponsors 
are  for  the  best  interests  of  all  concerned.  Much  criticism,  both  just 
and  unjust,  has  been  directed  to  those  responsible  for  the  standard 
methods.  It  would  be  well,  then,  to  study  the  reasons  underlying  the 
procedures  which  are  suggested  by  these  committees.  Usually  this 
criticism  is  directed  toward  the  medium.  Some  laboratory  worker, 
frequently  unversed  in  the  methods  of  research,  collects  the  medium 
from  several  other  laboratories  and  determines  the  colony  count  of  a 
given  milk  sample  upon  these  various  media.  His  results  show  varia- 
tions and  immediately  he  assumes  that  something  is  wrong.  It  is. 
The  report  of  the  committee  appointed  by  the  American  Public  Health 
Association  upon  the  media  in  actual  use  by  various  laboratories  shows 
alarming  and  absolutely  unnecessary  variations.  Why,  then,  should 
absolute  concord  be  expected  when  a  choice  of  several  peptones  is 
made  and  when  the  amounts  of  all  the  ingredients  used  vary  between 
wide  limits?  These  criticisms  of  the  medium  are  unjust — unjust 
until  the  real  standard  medium  is  used  universally.  Not  only  is  this 
statement  true,  but  there  are  other  elements  which  also  enter  into 
these  variations  which  in  turn  are  ascribed  to  the  medium.  Two  of 
these  are  the  pipettes  used  and  the  dilution  methods,  which  will  be 
elaborated  upon  in  their  proper  sequence. 

The  purpose  of  this  circular  is  to  describe  not  only  the  methods 
used  in  the  laboratory,  but  to  evaluate  the  relative  merits  of  the 
various  methods  which  are  used  in  the  enumeration  of  bacteria  in 
milk.  It  is  written  more  especially  for  the  plant  man  or  the  large 
dairyman,  since  the  workers  in  the  control  laboratory  of  the  city  and 
state  are  usually  previously  trained  to  do  this  work. 


PLATE    METHOD 

The  plate  method  is  essentially  a  device  to  trap  bacteria  in  a  thick 
jelly-like  substance.  In  this  substance  the  bacteria,  not  being  able 
to  move,  develop  with  amazing  rapidity  in  the  place  where  they  are 
trapped.  This  makes  their  progeny,  all  massed  together  in  a  single 
spot  (the  colony),  visible  to  the  naked  eye,  whereas  the  original  "seed- 
ing" organism  would  pass  unseen.  Sterile  apparatus,  carefully 
measured  dilutions,  and  cleanly  methods  in  carrying  out  the  operation 
are  required. 

There  are  nine  points  or  details  of  the  method  which  are  quite 
necessary  to  bear  in  mind  all  of  the  time  when  one  is  held  responsible 
for  the  plating  of  milk  for  bacteria. 


4  UNIVERSITY    OF    CALIFORNIA — EXPERIMENT    STATION 

These  nine  points,  which  will  be  discussed  point  by  point,  are: 
(1)  the  laboratory,  (2)  the  equipment,  (3)  collection  of  samples,  (4) 
transportation  of  samples  to  the  laboratory,  (5)  the  medium,  (6) 
plating,  (7)  incubation,  (8)  counting,  and  (9)  reporting. 

The  Laboratory. — A  laboratory  may  be  either  white-tiled  through- 
out, equipped  with  polished  metal  autoclaves  and  stills,  or  it  may  be 
any  room  set  apart  from  the  rest  of  the  plant  for  the  sole  purpose  of 
controlling  the  quality  of  the  incoming  milk  and  of  the  efficiency  of 


Incubator 


Scales 


'Store 


Sterilizer 


<4utoc/are 


O 


Fig.  1. — Plan  of  laboratory  for  a  plant  where  both  bacteriological  and 
fat  tests  are  to  be  made. 


the  equipment  used  in  the  plant.  The  outlay  for  installation  depends 
on  the  point  of  view  of  the  management.  The  laboratory  may  be  of 
the  nature  of  a  much  advertised  display,  or  an  unassuming  work  room. 
The  display  idea  is  excellent,  since  it  brings  to  the  attention  of  the 
public  the  thought  that  the  distributor  is  attempting  to  protect  the 
quality  of  his  product  and  the  well-being  of  his  patrons.  Such  a 
laboratory  would,  however,  be  expensive  to  install  and  maintain. 

Most  plant  laboratories  are  small,  clean  rooms,  having  perhaps  a 
minimum  of  equipment,  but  nevertheless  carrying  out  the  funda- 
mental purpose  of  the  laboratory  control.     It  should  be  clean,  light, 


CIRC.   310]  BACTERIOLOGICAL   LABORATORY   FOR   DAIRY   PLANTS  5 

accessible,  and  dry,  should  be  large  enough  for  the  operator  to  work 
with  ease,  and  should  have  a  north  light  if  microscopic  work  is  to  be 
undertaken. 

A  room  12  feet  by  14  feet  seems  about  the  right  size.  Anything 
smaller  would  handicap  the  analyst  and  the  heat  from  autoclaves  and 
sterilizers  would  be  unpleasant.  Figure  1  is  a  plan  for  a  laboratory 
which  is  not  far  from  ideal.  The  room  has  windows  on  two  sides, 
one  side  being  to  the  north.  Since  the  work  of  the  one  in  the  labora- 
tory usually  includes  the  Babcock  test,  this  too  is  indicated.  All  of 
the  apparatus  that  requires  the  use  of  steam  or  gives  out  much  heat 
has  been  placed  in  one  end  of  the  room.  Near  the  Babcock  tester  is 
the  sink,  with  the  stove  and  media-making  apparatus  beyond.  The 
incubator  is  indicated  as  being  below  the  table  top  in  the  corner.  The 
plates  are  prepared  on  the  table  along  the  north  wall,  and  are  counted 
there.  No  space  for  an  ice  box  is  shown  because  it  is  assumed  that 
media  will  be  kept  in  the  cold  rooms  of  the  plant. 

Electric  flush  receptacles  should  be  generously  installed  at  the 
time  the  laboratory  is  being  finished.  Table  tops  should  be  stained 
with  aniline  black.    To  do  this,  two  solutions  are  made: 

Solution  A — 

Aniline     120  grams 

HC1  (commercial)   180  grams 

Water  1000  cc. 

Solution  B — 

Sodium  dicliromate  120  grams 

Hydrochloric  acid  100  grams 

Water    1000  cc. 

Solution  A  should  be  applied  with  a  brush  to  the  fresh  smooth  surface 
and  allowed  to  dry  overnight.  The  color  will  turn  bright  yellow. 
Solution  B  should  then  be  spread  on  the  wood,  which  will  turn  dark 
and  be  very  streaky  at  first.  After  this  second  coat  dries  the  surface 
should  be  rubbed  with  vaseline,  motor  oil,  or  paraffine.  Vaseline 
seems  to  be  preferable. 

In  a  dairy  plant  where  there  is  plenty  of  steam,  a  connection 
should  be  made  with  the  plant  supply.  Running  water  and  electricity 
are  essential.  Gas  is  a  desirable  convenience,  but  can  be  replaced  by 
electricity  if  not  available. 

Equipment. — Below  is  given  a  list  of  the  articles  that  are  needed, 
together  with  the  approximate  cost.  With  this  equipment  properly 
set  up  an  operator  can  do  consistently  good  work  if  he  understand  the 
procedure. 


UNIVERSITY    OF    CALIFORNIA EXPERIMENT    STATION 


Article  Number 

Autoclave  (pressure  cooker)  

Stove    (2-3    burner)  

Double  boiler  

Sterilizer    (hot-air)    

Incubator,   electric   

Scales  

Weights,  1  gram— 100  grams 

Funnels,  6"  2 

Funnels,  8"  2 

Funnels,  3"  6 

Bottles    (6-oz.   prescription   ovals)  1  gross 

Bottles    (2-oz.   prescription   ovals)  1  gross 

Graduates,  1000  cc 1 

Graduates,  500  cc 2 

Graduates,  100  cc 3 

Flasks,  1000  cc.  Pyrex 24 

Flasks,  200  cc 36 

Petri  dishes,  100  X  15  mm n*  X  15 

Pipettes,  1  cc n3  X  15 

Pipettes,    100   cc 2 

Pipettes,   10    cc 10 

Pipette  box   2 

Counting    lens 1 

Counting  plate  1 

Counting  hand  tally 1 

Test   tubes,   6"  X  %" 1  gross 

Thermometer,  10°  to  100°  C 2 

Thermometer,  0°  to  250°  C 1 

Microscope    1 

Breed  pipette  1 

Microscopic  slides  1  gross 


Cost  per  unit 
$35.00 

6.00 

2.00 
45.00 
80.00 

9.00 

2.00 

0.30 

0.40 

0.10 

4.50 

3.50 

2.00 

0.75 

0.35 

0.50 

0.30 

0.18 

0.18 

0.25 

0.20 

3.00 

3.00 

0.15 

5.00 

2.80 

2.20 

1.50 
125.00 

1.25 

1.35 


To  this  list  should  be  addd  as  property  constantly  being  replaced : 

Article  Number  Cost  per  unit 

Difco    peptone    lib.  $6.00 

Filter  paper  1  package  1.00 

Cotton  absorbent,   cotton  batting,  pinch  cocks,   rubber  tubing,  corks,   etc., 

cork  stoppers,  wax  pencils,  pH  indicator  brom  thymol  blue,  dropping 

bottles,  stains. 


The  equipment  should  include  a  steam  sterilizer,  which  may  be 
one  of  the  autoclaves  listed  in  the  catalogues,  or  in  a  small  laboratory 
it  may  be  a  pressure  cooker  such  as  the  housewife  has  learned  to  use 
in  the  kitchen.  If  a  pressure  cooker  is  used,  it  should  be  fitted  for 
steam  by  the  shop  mechanic  to  hasten  the  sterilization  process. 


3 'n*  in  this  case  refers  to   the  number   of   samples  to  be  plated   daily, 
samples  daily,  6  X  15  =  90  plates  needed  to  keep  the  work  going. 


Six 


ClRC.   310]         BACTERIOLOGICAL  LABORATORY   FOR   DAIRY   PLANTS  7 

A  dry  sterilizer  for  glassware  and  an  incubator  are  essential.  For 
a  hot-air  sterilizer  it  is  possible  to  utilize  the  common  tin  oven  which 
can  be  purchased  for  a  few  dollars  from  the  hardware  stores.  A 
wooden  box  can  be  used  for  an  incubator  if  a  thermostat  is  purchased, 
but  it  would  be  best  to  buy  these  pieces  complete,  since  the  equipment 
on  the  market  is  apt  to  be  better  and  more  efficient  than  makeshift 
appliances.  However,  if  the  plant  has  in  its  employ  one  who  can 
construct  such  things  it  is  possible  to  save  some  money. 

Such  an  array  of  chemical  laboratory  equipment  might  confuse 
the  uninitiated,  and  the  uses  to  which  some  of  these  are  put  would 
be  difficult  of  description,  so  those  to  whom  these  articles  are  new 
would  do  well  to  visit  some  nearby  health  laboratory  to  familiarize 
themselves  with  their  appearance  and  use.  It  is  assumed  that  the 
reader  has  some  knowledge  of  laboratory  procedure. 

The  petri  dishes  should  be  100  mm.  outside  diameter  with  prefer- 
ably a  15  mm.  side  wall.  Pyrex  glass  is  expensive,  but  where  it  can 
be  procured  it  would  be  worth  the  price  paid  because  it  lessens  the 
breakage  bill,  which  is  high  in  most  laboratories.  The  use  of  clay 
tops,  in  vogue  ten  years  ago,  is  finding  less  support  today.  Not  only 
do  the  films  of  media  dry  out  more  rapidly,  but  the  ease  of  observing 
the  dishes  during  incubation  and  during  the  mixing  of  the  agar 
dilution  is  lost. 

In  sterilization,  the  plates  and  pipettes  should  be  placed  in  metal 
containers.  A  container  9"  X  9"  X  9"  is  a  convenient  size,  since  it 
will  permit  four  piles  of  plates,  10  to  a  pile.  Unless  plates  are  steril- 
ized by  the  methods  described  there  is  danger  of  subsequent  contam- 
ination through  air.  The  plate  should  be  kept  in  these  containers 
until  used. 

The  straight-sided  pipettes  are  best  for  bacteriological  use  since 
they  are  more  easily  placed  in  the  containers.  Right  here  is  where 
the  first  chance  for  inaccuracy  of  plate  counts  is  encountered.  It  has 
been  found  that  the  1  cc.  pipettes,  as  purchased  on  the  market,  fre- 
quently are  inaccurate  to  the  extent  of  10  per  cent  or  more.  To  guard 
against  errors  of  this  sort,  each  pipette  should  be  filled  with  distilled 
water  to  the  mark,  and  this  water  is  weighed.  It  should  weigh 
one  gram ;  if  it  is  off  as  much  as  10  milligrams  in  either  direction, 
the  pipette  should  be  discarded. 

Another  source  of  error  is  broken-tipped  pipettes.  The  pipettes 
in  general  use  today  are  made  to  deliver  1  cc,  that  is,  there  is  but 
one  mark  upon  the  pipette,  up  to  which  the  liquid  to  be  measured 
is  drawn.     After  drawing  to  this  mark  the  liquid  is  allowed  to  flow 


8  UNIVERSITY    OF    CALIFORNIA — EXPERIMENT    STATION 

out  by  gravity.  It  is  obvious,  then,  that  should  the  tip  of  the  pipette 
be  broken  the  pipette  is  useless  where  accurate  amounts  are  to  be 
delivered.  The  constant  use  to  which  these  pipettes  are  put,  the  oper- 
ations of  cleaning  and  sterilizing,  and  the  shaking  about  while  in  the 
case,  all  tend  to  increase  the  liability  of  such  breakage.  The  best 
way  to  overcome  this  source  of  inaccuracy  is  to  use  pipettes  made  to 
contain  1  cc,  which  have  two  marks,  one  well  up  on  the  pipette,  the 
other  near  the  tip.  These  pipettes  are  used  in  the  same  manner  as 
the  ones  in  general  use  with  the  exception  that  instead  of  allowing 
the  liquid  to  flow  out  completely,  it  is  allowed  to  run  down  to  the 
lower  mark.     Any  breakage  of  the  tip  with  this  pipette  is  of  little 


Fig.  2. — Types  of  closures  used  for  dilution  bottles.  The  cotton  plug  is  no 
longer  much  in  favor.  The  wine  cap  (the  center  two)  is  used  much  in  California 
and  has  many  advocates.  The  screw-top  bottle  is  used  in  this  laboratory  where 
it  has  proved  of  great  help. 

consequence  as  far  as  the  accuracy  of  the  volume  of  the  liquid  de- 
livered is  concerned.  Such  pipettes  cost  but  little  more  than  the  usual 
types. 

Dilution  bottles  next  claim  attention.  In  figure  2  are  shown 
various  types  of  dilution  bottles  and  of  closures.  The  use  of  the 
cotton  plug  is  to  be  discouraged,  because  the  plug  gets  wet.  Some 
laboratories  use  a  rubber  stopper  and  others  the  crown  seal  such  as 
is  found  on  soda  bottles.  This  laboratory  has  tried  many  of  the 
methods  used  to  seal  the  dilution  bottles  and  has  adopted  the  screw 
finish  bottle.  The  seals  come  in  different  sizes  for  the  two  sizes  of 
bottles,  so  a  yellow  seal  is  used  on  the  larger  bottle,  while  a  red  one 
fits  the  smaller.  Figure  2  also  shows  a  type  of  closure  very  popular 
in  laboratories  in  California.     Wine  caps  such  as  were  used  on  wine 


CIRC.   310]  BACTERIOLOGICAL   LABORATORY   FOR  DAIRY   PLANTS  9 

bottles  in  former  days  are  admirable  for  closing  dilution  bottle;  the 
chief  objection  is  that  the  dilution  water  tends  to  leak  out  past  the 
thumb  which  is  used  to  hold  the  cap  in  place.  Those  workers  favoring 
the  wine  cap  claim,  however,  that  this  is  no  disadvantage. 

The  dilution-bottle  method  offers  another  source  of  error  unless 
reasonable  care  is  used  in  measuring  the  water.  The  use  of  graduates 
for  this  work  is  to  be  deplored.  A  100  cubic  centimeter  pipette  is 
often  used.  In  doing  this,  however,  the  100  cc.  level  is  marked  on 
the  lower  end  of  the  pipette.  In  use,  the  pipette  is  inverted  and  the 
water  is  drawn  up  easily  through  the  larger  opening. 

Automatic  delivery  pipettes  in  which  one  side  of  the  apparatus 
fills  while  the  other  empties  are  much  better,  although  their  action  is 
sometimes  slow. 

In  sterilizing  the  glassware  dry  heat  should  be  used.  The  standard 
method  calls  for  175°  C  (347°  F)  for  one  hour,  but  such  a  time  and 
temperature  would  leave  one  in  doubt  as  to  absolute  sterility.  A 
longer  time  at  a  little  higher  temperature,  say  two  hours  at  190°  C, 
would  be  safer.  This  is  the  practice  in  the  laboratory  of  the  Dairy 
Industry  Division.  The  dilution  bottles  should  be  sterilized  in  the 
autoclave  as  should  the  medium. 

Collection  of  Samples. — In  the  collection  of  samples  too  much  care 
cannot  be  exercised,  although  too  little  attention  is  usually  given  to 
this  operation.  It  is  the  connecting  link  between  the  milk  to  be  exam- 
ined and  the  laboratory. 

In  gathering  samples  a  three-ounce  screw-top  bottle  (see  fig.  2) 
is  best,  although  cotton-plugged  tubes  are  still  found  in  many 
laboratories.  For  taking  the  sample  from  the  containers,  a  glass  or 
aluminum  tube  is  used.  The  tube  is  usually  about  25-30  inches  long 
with  an  inside  diameter  of  a  quarter  inch.  The  relative  merits  of 
glass  and  metal  tubes  are  more  or  less  obvious.  Glass  tubes  show 
whether  they  are  clean  or  not,  but  they  are  prone  to  break.  The 
question  also  arises  whether  a  slender  tube  is  capable  of  thoroughly 
stirring  the  contents  of  a  can.  Experiments  conducted  in  this  labora- 
tory indicate  that  representative  samples  can  be  taken  by  this  means. 

The  sample  should  be  placed  in  ice  unless  it  Is  to  be  plated  at  once. 
Bottled  milk  should  be  shaken  and  sampled  after  very  careful  removal 
of  the  cap,  during  which  operation  the  instrument  used  for  opening 
should  not  pierce  the  lower  surface. 

The  Medium. — Before  the  samples  reach  the  laboratory  a  supply 
of  the  medium  which  is  to  be  used  to  grow  the  bacteria  should  be  on 
hand. 


10  UNIVERSITY    OF    CALIFORNIA — EXPERIMENT    STATION 

The  standard  medium  has  the  following-  composition: 

Agar 15  grams 

Peptone 5  grams 

Meat  extract 3  grams 

Water 1000  cc. 

pH 6.8-7.0 

Distilled  water  should  be  used  because  it  is  more  constant  in  its 
composition  than  tap  water.  In  this  should  be  dissolved  the  peptone, 
which  is  a  soluble  "predigested"  meat  product.  The  meat  stuff  from 
which  it  is  prepared  is  composed  of  protein,  which  for  the  most  part 
is  insoluble.  By  digesting  the  meat  with  steam  in  an  acid  solution, 
the  meat  is  broken  down  into  its  component  parts:  amino  acids,  pro- 
teoses, and  peptones,  hence  the  name.  There  are  several  peptones  on 
the  market,  all  of  which  are  permitted  in  the  preparation  of  the 
medium.  It  would  seem  that  a  better  procedure  would  be  to  designate 
a  single  brand  of  peptone,  rather  than  allow  any  one  to  be  used. 
There  are  undoubtedly  differences  in  brands  and  in  their  ability  to 
support  growth.  Perhaps  a  way  out  of  this  difficulty  would  be  to 
designate  their  composition  or  the  stuff  from  which  they  are  made. 

In  the  water  is  also  dissolved  a  product  called  beef  extract,  which 
is  the  water  extract  of  ground-up  meat  from  which  the  water  has  been 
removed.  Both  of  these  ingredients,  although  composed  of  a  varying 
mass  of  nitrogenous  substances,  nowadays  are  controlled  in  their 
manufacture  so  that  a  given  brand  is  constant  in  its  composition. 

Agar  is  next  added  to  the  water.  Agar  does  not  go  into  true 
solution  but  swells  up  and  loses  its  shred-like  structure,  giving  the 
medium  a  slightly  cloudy  appearance. 

After  all  of  these  have  been  added  to  the  water,  the  hydrogen-ion 
concentration  is  determined.  The  determination  of  the  hydrogen-ion 
concentration  is  not  a  difficult  operation  when  once  understood.  It  is 
somewhat  different  from  the  titrametric  method  (the  phenolphthalein 
acid,  test)  in  which  a  single  indicator  is  used  and  acid  or  alkali 
added  to  bring  the  color  of  the  solution  to  the  'neutral'  point  of  the 
indicator.  In  the  determination  of  the  hydrogen-ion  concentration 
several  indicators  may  be  used,  all  showing  color  changes  but  at 
diffent  acidities.  Without  going  into  the  basic  theories  of  the  reactions 
involved  it  can  be  well  said  that  the  idea  is  to  determine  the  weight 
of  hydrogen  ions  in  a  liter  of  water  or  solution.  The  thermometer, 
as  is  well  known,  is  graduated  in  parts  which  are  known  as  degrees. 
In  a  similar  way  the  acid-alkali  scale  is  graduated,  so  to  speak,  in 
degrees  or  in  pH  values.  There  are  fourteen  of  these,  from  pII-0, 
which  is  a  solution  of  normal  acid  (36.5  grams  of  concentrated  HC1 


ClRC.   310]  BACTERIOLOGICAL   LABORATORY    FOR   DAIRY   PLANTS  11 

in  1000  cc.  water),  through  pH  7  or  the  "neutral"  point,  to  pH  14 
or  normal  alkali  (40  grams  sodium  hydroxide  in  1000  cc.  water). 

It  was  mentioned  above  that  for  the  colorimetric  determination  of 
hydrogen-ion  concentration  several  indicators  are  used.  Each  of  these 
indicators  displays  two  colors — one  for  extreme  acid  and  one  for 
extreme  alkali,  say  yellow  for  the  acid  and  red  for  the  alkali.  There 
is  some  place  in  the  pH  scale  where  the  transition  from  yellow  to  red 
takes  place  for  each  indicator.  In  this  transition  the  intermediate 
blending  of  the  yellow  to  the  red  colors  make  varying  degrees  of 
orange.  Indicators  have  been  selected  which  have  these  sensitive 
transition  zones  at  various  points  of  the  pH  scale  from  pH  0  to  pH  14. 
Figure  3  is  given  in  an  attempt  to  make  the  above  statement  clearer. 
In  this  figure  the  pH  values  are  represented  on  the  right.  The  vertical 
areas  numbered  1  to  7  represent  a  hypothetical  series  of  indicators. 
Each  of  these  indicators  is  represented  as  having  a  yellow  color  for 
extreme  acid  (Y  at  top)  and  red  for  extreme  alkali  (R  at  bottom). 
These  areas  represent  the  sensitive  zone  where  the  indicator  passes 
through  changing  orange  colors  from  yellow  to  red.  It  will  be  seen 
that  these  zones  overlap.  In  a  solution  of  pH  0  (fig.  3)  indicator  1 
would  be  yellow.  .All  others  would  be  yellow  as  well.  If  now  we 
should  add  alkali,  a  drop  at  a  time,  to  this  solution,  indicator  1  would 
slowly  change  to  red  as  the  pH  became  greater,  or  as  the  solution 
became  more  alkaline.  But  before  the  full  red  color  of  indicator  1 
develops  indicator  2  would  begin  to  change  to  red.  This  will  be  true 
throughout  the  pH  scale,  and  demonstrates  the  general  statement  that 
every  pH  has  some  indicator  which  displays  a  telltale  color  by  which 
that  pH  can  be  determined. 

With  this  explanation  in  mind,  let  us  assume  that  we  have  a 
solution  A  which  is  pH-0.5.  Indicator  1  will  be  yellow  with  a  slight 
trace  of  red  in  it,  a  very  yellowish  orange.  Every  other  indicator 
will  be  wholly  yellow.  Taking  in  order  indicators  B,  C,  D,  etc.,  table  1 
gives  the  color  each  indicator  will  be  at  that  pH. 

In  actual  practice  we  do  not  have  all  indicators  of  the  yellow-red 
variety,  nor  do  we  use  the  whole  range  pH  0  to  pH  14.  In  biology, 
in  general,  and  in  bacteriology,  in  particular,  the  range  is  usually 
from  pH  4.0  to  pH  9.0  and  we  use  about  4  indicators  to  cover  the 
range.  To  narrow  it  down  still  more,  the  media-maker  is  concerned 
with  but  one  indicator  and  its  color  changes.  This  is  represented  by 
indicator  8  in  figure  3,  or  Brom  thymol  blue  which  is  yellow  at  pli  6 
and  blue  at  pH  7.5,  with  all  shades  of  green  between.  To  aid  the 
worker  unfamiliar  with  such  things  an  artifice  is  employed  which 
closely  accords  with  absolute  values  known  to  more  exact  work. 


12 


UNIVERSITY    OF    CALIFORNIA — EXPERIMENT    STATION 

\o 


D  — 


fellow 


Yellow 


ted 


Y 


Red 


Yellow 


■Meufral  point 
Y 


Blue 


Y 


Y 


I 

a 

3 
A 
5 
6 

7\ 

S 

9 

10 

II 

IE 

13 
14 


Fig.  3. — The  theory  of  the  use  of  several  indicators.     Each  indicator  changes 
from  its  full  acid  color  to  its  full  alkaline  color  at  definite  narrow  pll  ranges. 

TABLE  1 

Colors  that  Various  Indicators  Shown  in  Figure  3  Will  Have  at 

Points  A-B,  etc. 


pHof 
solution 

Indicator 

Point 

1 

2 

3 

4 

5 

6 

7 

will  be 

B 

1.6 

Red- 

Yellow 

Yellow 

Yellow 

Yellow 

Yellow 

Yellow 

C 

4.7 

orange 
Red 

Red 

Yellow- 

Yellow 

Yellow 

Yellow 

Yellow 

D 

6.7 

Red 

Red 

orange 
Red 

Yellow- 

Yellow 

Yellow 

Yellow 

E 

8.7 

Red 

Red 

Red 

orange 
Red 

Yellow- 

Yellow 

Yellow 

F 
G 

10.7 
12.7 

Red 
Red 

Red 
Red 

Red 
Red 

Red 
Red 

orange 
Red 
Red 

Orange 
Orange 

Yellow 
Orange 

CIRC.   310]         BACTERIOLOGICAL   LABORATORY    FOR   DAIRY   PLANTS 


13 


A  series  of  six  tubes  is  placed  in  a  test  tube  rack  and  to  these  tubes 
is  added  a  weak  solution  of  hydrochloric  acid  (1  drop  of  acid  to  100  cc. 
of  water)  5  cc.  to  a  tube.  Another  set  of  six  tubes  is  placed  in  front 
of  these  into  which  is  placed  5  cc.  of  0.5  per  cent  sodium  hydroxide 
solution.  To  one  tube  of  the  acid  is  added  9  drops  of  the  indicator 
Brom  thymol  blue,  to  the  next  8  drops,  then  7  drops,  etc.  It  will  be 
seen  that  there  is  a  decreasing  intensity  of  the  yellow  color  as  less 
and  less  of  the  indicator  is  used.  Now  to  the  series  of  alkali  tubes  1 
drop  of  indicator  is  added  to  the  first  tube,  two  to  the  second  and  so 
on,  which  gives  an  increasing  intensity  of  blue  as  more  of  the  indicator 
is  added. 

A  B  C  D  E  r 


Acid 
Tubes 


I 

0^ 


I 


'a 


i 

<0 


\ 


Alkali 

Tubas 


! 


(VI 


! 


E. 


I 


a. 


I 


pH  6.2  6.4         6.7         6.9  7.1  7.J 

Fig.  4. — The  drop-ratio  method  of  determining  pH  values. 


When  this  is  done  there  will  be  six  pairs  of  tubes,  each  pair  will 
have  10  drops  of  indicator  between  them  (9  plus  1,  or  8  plus  2,  etc.). 
It  so  happens  that  on  looking  through  any  pair  of  tubes  the  shades 
of  green  will  correspond  to  certain  pH  values. 

Figure  4  brings  this  out  more  clearly.  The  pair  of  tubes  marked  A 
(reading  down)  having  between  them  10  drops  of  indicator  will  have 
a  color  corresponding  to  pH  6.2  when  they  are  observed  one  behind 
the  other.  To  better  perform  this  operation  a  comparator  block  is 
used  which  is  shown  in  figure  5.  The  tubes  are  inserted  and  observed 
through  the  holes  bored  in  the  side  of  the  block.    Figure  6  shows  the 


14  UNIVERSITY    OF    CALIFORNIA EXPERIMENT    STATION 

block  ready  for  use.  In  this  figure  the  six  pairs  of  tubes  described 
above  are  shown,  one  pair,  however,  is  in  the  comparator  block. 
Behind  this  pair  is  placed  a  tube  of  medium  since  it  normally  has  a 
color  of  its  own  which  must  be  taken  into  consideration  in  matching. 
In  the  block  is  also  placed  a  tube  containing  1  cc.  of  the  medium,  4  cc. 
of  water  and  10  drops  of  indicator.  Behind  this  there  are  frequently 
placed  two  tubes  of  water,  although  this  is  not  necessary. 

If  the  color  of  the  indicator  in  the  medium  does  not  correspond  to 
the  color  of  the  tubes  between  pH  6.6  to  pH  7.0  adjustment  is  neces- 
sary.    To  adjust  the  medium  N/204  NaOH  is  added  from  a  burette 


Fig.  5. — The  comparator  block. 

or  pipette  into  the  tube  until  the  desired  shade  of  green  is  obtained. 
Fifty  times  this  amount  of  N  NaOH  is  now  added  to  a  liter  of  the 
medium  which  will  be  found  to  be  of  the  desired  pH. 

The  medium  is  now  placed  in  flasks  or  bottles  in  100  cc.  quantities, 
and  sterilized  in  the  autoclave  for  25  minutes  at  15  pounds  pressure. 

When  time  is  at  a  premium  dehydrated  media  can  be  used.  This 
is  a  complete  medium  with  the  water  extracted.  All  that  is  necessary 
for  preparing  a  liter  of  standard  agar  is  to  weigh  out  the  required 
amount  of  the  dry  powder,  dissolve,  and  sterilize.  The  initial  cost  of 
this  medium  is  higher  than  that  prepared  as  given  above.     In  a  busj' 

*  N/20  NaOH  is  made  by  dissolving  2  grams  of  sodium  hydroxide  in  1000  cc. 
of  water.  N  NaOH  is  20  times  stronger.  For  such  work  as  described  above  these 
solutions  would  not  have  to  be  corrected,  although  correction  would  be  necessary 
if  refined  work  were  done. 


CIRC.   310]  BACTERIOLOGICAL    LABORATORY    FOR   DAIRY    PLANTS 


15 


plant  laboratory  where  the  operator  is  required  to  do  work  other  than 
that  of  a  strict  bacteriological  nature,  this  extra  cost  would  not  be  a 
factor  of  importance. 

A  method  nearly  as  rapid  is  recommended  by  standard  methods. 
A  concentrated  agar  solution  is  made.  This  is  melted  when  agar  is 
needed,  and  a  concentrated  solution  of  the  peptone  and  extract  is 
added  to  it,  For  this,  the  agar  for  1  liter  is  dissolved  in  600  cc.  of 
water  while  the  nutrient  (peptones,  etc.)  for  the  liter  is  dissolved  in 
400  cc.  of  water.  The  agar,  however,  is  made  up  in  large  quantities 
and  stored  in  600  cc.  amounts.  Whenever  a  liter  is  needed  the  nutrient 
solution  is  made  up  and  added  to  the  agar. 


Fig.  6. — The  color  standards  and  comparator  block  in  use. 

Sometimes  for  special  work  in  bacteriology,  sugars  and  other 
fermentable  substances  are  used  for  diagnosis,  and  aiding  in  the 
growth  of  the  colonies.  Lactose  is  of  great  importance,  especially 
in  studying  milk  bacteria.  However,  although  there  are  some  who 
believe  that  the  medium  would  be  greatly  benefitted  by  such  an 
addition,  it  is  not  standard  practice. 

Plating.— On  arrival  at  the  laboratory  of  the  samples  to  be  tested 
the  actual  work  of  plating  should  be  started  at  once,  especially  if 
the  samples  mentioned  are  to  be  the  only  lot  received  that  day. 
Should  there  be  several  inspectors  collecting  samples,  as  is  the  case 
in  large  city  laboratories,  it  is  permissible  to  wait  for  all  samples  to 
be  brought  to  the  laboratory,  provided  they  are  kept  at  a  temperature 
below  40°  F.  The  samples  must  be  completely  covered  with  ice  in  a 
closed  container  to  bring  them  down  to  that  temperature. 


16  UNIVERSITY    OF    CALIFORNIA EXPERIMENT    STATION 

Having  then  sterilized  all  equipment,  and  with  the  samples  to  be 
plated  at  hand,  the  analyst  is  ready  to  begin.  The  table  top  should 
be  thoroughly  wiped  with  a  damp  cloth  to  remove  the  dust  that  may 
have  collected  there.  The  plates,  dilution  bottles,  and  samples  should 
be  carefully  arranged  along  the  table.  First  from  the  edge  should  be 
placed  the  petri  dishes;  next  the  sample  to  be  plated,  and  lastly  the 
dilution  bottles.  The  petri  dishes  should  then  be  marked  with  sample 
numbers,  date,  and  dilution.  This  laboratory  has  used  a  method  of 
marking  the  petri  dishes  which  is  worthy  of  mention.  Instead  of  the 
long  rows  of  ciphers  it  would  be  necessary  to  write  down  if  a  plate 
were  marked  "  100,000' '  or  "1,000,000"  the  following  shorter,  more 
easily  read  system  is  used:  100  becomes  H,  1,000  becomes  T,  10,000 
becomes  0T,  100,000  becomes  00T,  1,000,000  becomes  M.  Also  one 
will  frequently  read  "100,000"  as  though  it  contained  one  cipher 
more  or  less. 

Assuming  everything  is  now  in  readiness,  the  plating  begins.  The 
theory  of  the  procedure  is  that,  since  it  would  be  impractical  to  count 
the  bacteria  in  a  whole  cubic  centimeter  of  milk,  a  fraction  of  a  cubic 
centimeter  is  used.  The  fraction,  1/1,000  or  1/1,000,000  of  a  cubic 
centimeter,  is  chosen  according  to  the  analyst's  estimate  of  the  prob- 
able germ  content  of  the  milk.  To  get  these  fractions  of  a  cubic 
centimeter  (cc),  the  milk  is  mixed  with  sterile  water.  One  could, 
of  course,  in  making  a  dilution  of  1-1,000,000,  take  a  single  cubic 
centimeter  and  add  it  to  1,000  liters  of  water  or  264  gallons  and  take 
a  sample  of  that.  Such  methods  would  be  absurd,  so  a  system  of 
progressive  decimal  dilutions  are  made.  In  so  doing,  taking  1  to 
1,000,000  dilution  again  as  an  example,  3  bottles  are  used,  each  con- 
taining 99  cc.  of  distilled  water.  One  cc.  of  milk  is  added  to  the  first 
99  cc.  of  water.  The  bacteria  in  this  1  cc.  of  milk  are  then  floating 
about  in  100  cc.  of  fluid  (1  part  milk  and  99  parts  water)  which  is 
called  the  "l-to-100  dilution."  If  this  in  turn  is  again  diluted  by 
adding  1  cc.  of  the  l-to-100  dilution  to  a  second  99  cc.  of  water  the 
bacteria  will  be  floating  in  a  fluid  composed  of  1  part  milk  and  9999 
parts  water  and  is  the  l-to-10,000  dilution.  This  can  continue  until 
several  99  cc.  portions  are  taken.  The  jump  from  a  dilution  of  1-100 
to  1-10,000  is  large,  so  it  is  the  practice  in  laboratories  to  use  a  9  cc. 
dilution  between  two  99  cc.  dilutions. 

Table  2  is  given  to  more  fully  explain  the  process  of  making  the 
dilutions.    The  first  99  cc.  dilution  receives  the  milk  from  the  sample. 

A  little  study  of  the  above  table  should  make  the  procedure  clear 
to  anyone.  It  is  not  necessary  to  make  the  first  9  cc.  dilutions  if 
dilutions  of  1-100,000  are  to  be  made  nor  the  second  9  cc.  dilution  if 


ClBC.   310]         BACTERIOLOGICAL   LABORATORY   FOR   DAIRY   PLANTS 


17 


1-1,000,000  are  desired.  It  is  wise  unless  one  is  certain  of  the  prob- 
able number  of  bacteria  in  a  sample  of  milk  to  make  at  least  two 
dilutions. 

TABLE  2 
Outline  of  Method  Used  in  Making  Dilutions 


Step 

Source 

Dilution  bottle 
used 

Resulting  dilution 

Mark  on 
Petri  dish 

A 

Sample 

99  cc 

1-100 

H 

B 

1-100  dilution 

9 

1-1000 

T 

C 

1-100  dilution 

99 

1-10,000 

OT 

D 

1-10,000  dilution 

9 

1-100,000 

OOT 

E 

1-10,000  dilution 

99 

1-1,000,000 

M 

The  standard  methods  require  that  the  original  sample,  as  well 
as  the  dilutions,  should  be  shaken  25  times  before  subsequent  portions 
are  removed.  The  reason  for  this  is  evident  when  it  is  known  that 
frequently  the  bacteria  in  milk  are  found  as  streptococci  and  strepto- 
bacilli,  in  which  chains  are  formed  containing  a  number  of  cells.  If 
the  dilution  is  made  without  shaking,  the  whole  chain  may  be  seeded 
into  the  agar  and  grow  into  a  colony  to  be  counted  as  one.  On  the 
other  hand,  if  the  dilution  is  shaken  excessively  the  chains  are  entirely 
broken  up,  causing  as  many  colonies  to  form  as  there  are  individuals 
in  the  chain.  Since  either  extreme  might  be  used,  and  since  excessive 
shaking  cannot  be  easily  denned,  a  middle  course  is  taken,  consisting 
of  shaking  25  times,  each  shake  being  an  up-and-down  motion  of 
about  one  foot.  Let  it  be  repeated  at  this  time  that  in  the  process 
of  plating,  sterile  glassware,  sterile  dilution  water,  and  sterile  media 
should  be  used  throughout. 

After  the  sample  is  shaken,  the  cap  should  be  removed  without 
piercing  the  paper,  and  one  cubic  centimeter  of  the  milk  removed. 
This  is  transferred  to  a  dilution  bottle  which  is  shaken  as  directed. 
The  pipette  should  not  be  blown  out;  the  fluid  should  be  allowed  to 
flow  out  of  its  own  accord.  It  should  not  be  rinsed  in  the  dilution. 
Dilutions  are  thus  made  until  one  is  reached  which,  in  the  opinion  of 
the  analyst,  is  correct  for  that  particular  sample.  One  cubic  centi- 
meter of  the  milk-and-water  dilution  is  then  transferred  to  a  petri 
dish  properly  marked. 

At  once,  or  within  20  minutes  at  the  most,  the  agar  is  poured  upon 
the  milk  dilution  and  the  two  mixed  thoroughly  with  a  gentle  rotary 
motion.     The  agar  is  then  allowed  to  solidify,  after  which  the  plates 


18  UNIVERSITY    OF    CALIFORNIA EXPERIMENT    STATION 

are  placed  in  the  incubator  in  an  inverted  position.  This  inverting 
of  the  plates  is  a  precaution  against  'spreaders.'  If  the  plate  is  placed 
agar-side  down,  the  drops  of  condensation  water  falling  on  the  plate 
might  cause  contamination  if  it  should  strike  a  colony.  By  inverting 
the  plates  the  water  that  does  condense  remains  where  it  formed. 

Incubation. — In  order  to  stimulate  the  growth  of  the  bacteria 
seeded  in  the  agar,  the  plates  are  incubated  at  37°  C.  The  time  for 
incubation  has  also  been  specified  by  the  American  Public  Health 
Association  as  48  hours  and  should  be  adhered  to  closely.  The  plates 
in  the  incubator  should  be  stacked  not  too  closely,  because  close  stack- 
ing leads  to  inaccuracies  in  the  final  count.  It  has  also  been  suggested 
that  a  fan  be  placed  in  large  incubator  rooms  to  keep  the  air  in 
motion.  The  purpose  of  this  is  not  to  create  a  draft,  but  simply  to 
overcome  stratification  of  air.  The  temperature  of  the  air  at  various 
places  about  the  incubator  should  be  determined.  The  incubation  of 
the  plates  is  one  of  the  most  important  steps. 

Counting  Plates. — At  the  end  of  the  incubation  period,  the  plates 
should  be  removed  to  the  laboratory  and  the  colonies  counted. 
Theoretically,  each  colony  results  from  the  growth  of  a  single  organ- 
ism. The  number  of  colonies  is  then  multiplied  by  the  dilution,  but, 
if  two  dilutions  have  been  made  the  one  is  chosen  that  contains  from 
30  to  300  colonies.  This  is  done  because  it  has  been  found  from 
experience  that  plates  with  less  than  thirty  colonies  give  results  that 
are  high,  and  when  there  are  more  than  300  colonies  to  a  plate  there 
is  apt  to  be  crowding.  Products  of  growth  from  one  colony  diffuse 
through  the  medium  and  frequently  prevent  bacteria  in  other  colonies 
from  growing.  This  sometimes  gives  rise  to  the  formation  of  "pin 
point"  colonies. 

In  counting  plates  a  dark  background  is  used  such  as  is  pictured, 
in  figure  7.  Some  laboratory  workers  prefer  a  highly  illuminated 
background  but  the  author  has  found  the  dark  background  very  satis- 
factory. The  counting  is  done  with  a  hand  lens  magnifying  2% 
diameters. 

Standard  Methods  of  Milk  Analysis  discusses  the  common  sources 
of  error  in  plate  counts  as  follows : 

"Agar  plate  'counts'  per  cc.  are  to  be  regarded  as  estimates  of 
numbers  rather  than  as  exact  counts,  since  only  a  portion  of  a  cubic 
centimeter  is  used  in  preparing  the  plates.  As  such  they  are  (like  all 
estimates)  subject  to  certain  well  known  and  recognized  errors  whose 
size  can  be  largely  controlled  by  the  care  taken  in  the  analysis. 
Among  these  errors  are:  (a)  Failure  of  some  of  the  bacteria  to  grow 
because  the  incubation  temperature  or  the  composition  or  reaction 


CIRC.   310]  BACTERIOLOGICAL    LABORATORY    FOR   DAIRY    PLANTS 


19 


of  the  medium  is  not  suitable.  (&)  Inaccuracies  in  measurement  of 
the  quantities  used,  (c)  Mistakes  in  counting,  recording  data,  com- 
puting results,  and  the  like,  (d)  Incomplete  sterilization  of  con- 
tamination of  the  plates,  dilution  waters,  etc.  The  possible  errors 
caused  by  these  things  makes  it  highly  important  for  all  routine 
laboratories  to  follow  carefully  a  standard  procedure. 

''Recent  investigations  make  it  clear  that  these  largely  controllable 
errors  are  not  so  likely  to  cause  misconceptions  of  the  accuracy  of  the 
results  as  are  the  errors  due  to  the  fact  that  bacteria  in  milk  usually 
cling  together  in  groups  of  from  two  to  many  hundreds  of  individuals. 
These  groups  are  only  partially  broken  apart  by  the  shaking  given  in 
preparing  the  dilutions,  so  that  at  best  the  counts  from  the  agar  plates 


Fig.   7. — The  counting  plate,  the  tally  counter,  and  the  magnifying  glass 
used  in  counting  colonies. 

represent  the  number  of  isolated  individuals  and  groups  of  two  or 
more  bacteria  that  exist  in  the  final  dilution  water.  Thus  the  colony 
counts  from  the  plates  are  always  much  smaller  than  the  total  number 
of  bacteria  present.  This  error  would  not  be  troublesome  if  the 
groups  were  of  constant  average  size,  but  the  best  information  avail- 
able shows  that  the  groups  in  ordinary  market  milk  commonly  vary 
in  size  so  that  they  contain  an  average  of  from  2  to  6  individual 
bacteria.  Some  samples  contain  groups  of  even  smaller  size  than  this, 
while  others,  such  as  those  bearing  long-chain  streptococci,  may  show 
groups  containing  an  average  of  25  or  even  more  individual  bacteria. 
The  irregularity  of  this  error  (whose  size  is  not  indicated  in  any  way 
by  the  appearance  of  the  plates)  should  be  kept  in  mind  in  interpret- 
ing the  results  obtained. 

"Because  of  the  fact  that  agar-plate  counts  only  represent  a 
fraction  of  the  total  number  of  bacteria  present,  they  should  not  be 
reported  as  showing  the  'number  of  bacteria  per  cc'     Accurately 


20  UNIVERSITY    OF    CALIFORNIA EXPERIMENT    STATION 

speaking,  the  counts  from  agar  plates  give  the  estimated  number  of 
colonies  that  would  have  developed  on  standard  agar  per  cc.  of  milk 
if  an  entire  cubic  centimeter  of  milk  had  been  used  for  inoculation. 
Because  this  statement  of  fact  is  cumbersome,  and  also  because  a 
certain  ratio  exists  in  each  case  between  the  colony  count  and  the  total 
number  of  bacteria,  it  has  become  a  common  practice  to  speak  of  the 
plate  counts  as  showing  the  number  of  bacteria  per  cc.  This  is  very 
confusing  now  that  microscopic  methods  of  counting  have  been  devel- 
oped which  permit  counts  of  the  actual  bacteria  to  be  made.  The 
most  frequent  ratio  between  agar-plate  counts  made  by  the  official 
plating  method  and  the  total  number  of  bacteria  present  has  been 
found  to  be  approximately  1  to  4. 

"It  is  therefore  recommended  that  all  agar  plate  counts  obtained 
by  the  standard  technique  shall  not  be  reported  in  the  form  '  2,000,000 
bacteria  per  cc. '  but  rather  as  follows :  '  Official  plate  count,  2,000,000. ' 
The  latter  form  of  expression  shall  be  considered  an  abbreviated 
method  of  saying:  'a  count  of  2,000,000  colonies  per  cc.  as  obtained 
by  standard  methods.'  Moreover,  analysts  shall  be  careful  to  avoid 
giving  a  fictitious  idea  of  the  accuracy  of  the  official  plate  count. 
There  is  ample  justification  for  thinking  it  sufficiently  accurate  to 
justify  drawing  conclusions  as  to  the  general  quality  of  a  given 
sample  of  milk,  and  when  a  series  of  samples  from  the  same  source 
are  examined  the  average  result  may  permit  much  more  specific  con- 
clusions to  be  drawn  with  confidence." 


THE    MICROSCOPIC   COUNT 

The  microscopic  count,  also  known  as  the  Breed  or  direct  count, 
is  the  method  sponsored  by  Dr.  R.  S.  Breed,  in  which  the  bacteria 
themselves  are  seen  directly  by  means  of  a  microscope.  The  method 
was  developed  owing  to  a  demand  on  the  part  of  milk-plant  operators 
for  a  more  rapid  method  than  the  incubated-plate  method.  There 
have  been  many  criticisms  of  the  plate  method.  These  criticisms 
usually  are  directed  to  the  facts  that  all  of  the  bacteria  do  not  grow 
on  the  media  used,  that  it  takes  48  hours  to  obtain  results,  that  the 
underlying  principle  of  colony  growth  from  a  single  organism  is  not 
always  true,  and  that  many  of  the  operations  are  subject  to  relatively 
great  personal  errors  on  the  part  of  the  analyst. 

The  plate  method,  regardless  of  the  possible  truth  of  the  above 
criticisms,  has  been  used  for  many  years  and  around  its  findings  has 
been  developed  a  certain  well-defined  conception  of  what  constitutes 
a  good  milk  from  the  bacteriological  point  of  view.     Any  method, 


ClRC.   310]  BACTERIOLOGICAL   LABORATORY   FOR   DAIRY   PLANTS  21 

whether  it  is  a  direct  enumeration  of  the  bacteria  seen  by  the  micro- 
scope or  whether  it  involves  such  growth  phenomena  as  reductase 
production,  must  obviously  be  correlated  to  the  older  concepts  that 
the  plate  method  has  developed. 

Anyone  can  make  a  smear  of  milk  on  a  glass  slide  and  see  the  bac- 
teria after  staining.  The  significance  of  the  numbers  of  bacteria 
seen  is  of  greater  importance.  They  must  be  thought  of  in  terms  of 
the  unit  of  measure,  the  cubic  centimeter. 

This  difficulty  was  obviated  by  controlling  the  possible  variable 
factors  that  enter  into  the  method.  A  definite  quantity  of  milk 
(0.01  cc.)  is  spread  over  a  definite  area  (1  sq.  cm.),  which  gives  a 
film  approximately  0.1  mm.  in  thickness.  This  film,  after  staining,  is 
examined  with  a  microscope  having  a  field  of  view  of  a  definite  size 
(0.205  mm.).  The  milk  film  observed  through  the  microscope  is 
1/300,000  of  a  cubic  centimeter.  From  the  number  of  bacteria  seen  in 
this  amount  of  milk  the  number  in  the  whole  cubic  centimeter  can 
be  easily  calculated. 

Pipettes  are  now  sold  which  are  made  to  deliver  0.01  cc.  of  milk. 
Care  should  be  exercised  in  obtaining  these,  only  those  being  chosen 
which  have  a  ground-off,  cone-shaped  tip.  The  drop  should  be  dis- 
charged cleanly  and  should  not  run  back  on  the  side  of  the  tip. 
These  pipettes  vary  somewhat  and  it  would  be  well  to  calibrate  them 
by  determining  the  weight  of  milk  contained  in  the  pipette.  It  should 
deliver  0.0103  gram. 

A  single  pipette  is  needed  for  the  work.  It  need  not  be  sterile 
but  should  be  flushed  out  after  each  sample  of  milk  by  rinsing  in 
warm  clear  water.  The  pipettes  when  not  in  use  should  be  kept  in 
a  glass-cleaning  solution  or  in  sulphuric  acid. 

The  slides  used  may  be  the  common  1"  X  3"  microscope  slide. 
If  these  are  used,  a  card  can  be  purchased  which  serves  as  a  guide 
in  the  spreading  of  the  milk.  Slides  somewhat  larger  in  size  but  with 
the  surface  ground  except  for  little  windows  of  the  proper  size,  are 
also  in  use.  This  may  be  more  easily  visualized  by  referring  to 
figure  8.  Figure  8  a  shows  a  microscope  slide  with  the  film  of  milk 
spread  over  1  sq.  cm.  Figure  8  b  shows  a  cross  section  along  line 
A-B  (fig.  8  a)  with  the  milk  film  represented.  The  microscope 
objective  is  just  above  the  film  over  that  portion  which  is  .shaded. 
This  shaded  portion  is  next  shown  (fig.  8  c)  as  a  little  pellet  or  button 
of  milk  0.205  mm.  in  diameter  and  0.1  mm.  thick.  This  button  is 
1/300,000  of  a  cubic  centimeter.  From  the  number  of  organisms  seen 
in  this  button  (5  in  this  case)  the  number  per  cubic  centimeter  can 
be  calculated  by  simple  multiplication. 


22 


UNIVERSITY    OF    CALIFORNIA EXPERIMENT    STATION 


The  steps  in  the  microscopic  method  are  as  follows : 

1.  The  milk  is  drawn  into  the  pipette  and  the  tip  of  the  pipette  is 

wiped  with  a  clean  cloth. 

2.  The  milk  is  blown  onto  a  glass  slide. 

3.  It  is  spread  over  an  area  of  a  square  centimeter  by  means  of  a 

sterile  wire. 

4.  It  is  dried,  care  being  taken  not  to  dry  too  quickly  because  such 

drying  causes  cracking. 

5.  The  slide  is  placed  for  one  minute  in  a  Coplin  jar  containing 

xylol  to  remove  the  fat  from  the  film.  Gasoline,  or  any  other 
fat  solvent,  may  be  used,  but  the  xylol  is  suggested  in  the 
standard  methods. 

6.  The  slide  is  drained  and  dried.    The  film  now  has  a  frosty  white 
appearance. 


Milk  film 
O.lmm  thick 


Objective 


O/oss    s/ide 


Fig.  8. — Top:  Slide  with  milk  film  spread  over  1  square  centimeter.  Middle: 
Side  view  showing  milk  film  magnified  with  objective.  The  shaded  area  is  the 
portion  of  milk  film  seen  at  any  one  time.  Bottom:  Greatly  magnified  portion  of 
film  showing  that  portion  of  film  seen  at  any  one  time.  It  is  1/300,000  of  a  cubic 
centimeter. 


CIRC.   310]  BACTERIOLOGICAL   LABORATORY   FOR   DAIRY   PLANTS  23 

7.  The  slide  is  then  placed  in  a  second  Coplin  jar  containing  90 

per  cent  grain  alcohol  or  denatured  alcohol. 

8.  It   is   next   placed   for   at  least   one   minute   in   a   Coplin   jar 

containing  methylene-blue  solution  prepared  as  follows : 
saturated  alcoholic  solution  of  methylene  blue,  30  cc.,5  0.01- 
per-cent  solution  of  caustic  potash,  100  cc.  The  film  is 
purposely  left  in  the  bath  for  a  long  time  to  overstain. 

9.  The  film  is  decolorized  by  again  placing  the  film  in  the  alcohol. 

The  alcohol  draws  the  stain  from  the  milk  more  rapidly  than 
it  is  drawn  from  the  bacteria,  thus  giving  a  contrasting 
picture. 
10.  The  film  is  dried  and  examined. 
Before  the  film  can  be  examined  with  any  assurance  that  the  find- 
ings will  be  comparable  to  our  previous  understanding  of  what 
bacterial  numbers  in  milk  means,  the  microscope  must  be  adjusted. 
For  those  to  whom  the  microscope  is  new,  a  brief  description  of 
its  parts  may  be  of  interest.  In  figure  10  is  shown  the  parts  of  a 
laboratory  microscope.  To  the  stand  is  attached  a  mirror,  a  con- 
denser, a  stage,  and  a  body.  The  body  has  a  nose  piece  with  usually 
three  objectives  (lens  near  the  object  to  be  viewed)  and  a  selection 
of  eyepieces,  only  one  of  which  is  in  use  at  one  time.  By  varying 
the  eyepieces  and  objectives  one  can  approach  the  required  size  of 
the  field,  0.205  mm.  The  final  adjustment  is  made  by  pulling  out 
the  draw  tube.  The  steps  in  the  calibration  of  the  microscope  are 
simple  to  one  who  understands  microscopy.  To  the  layman,  however, 
it  would  be  difficult  to  explain,  and  explanation  is,  indeed,  unneces- 
sary, since  in  the  purchase  of  a  microscope  the  buyer  can  request  the 
desired  calibration  of  the  seller.  A  mechanical  stage,  which  is  an 
attachment  permitting  very  slow  and  controlled  movements  of  the 
slide  upon  the  stage,  is  an  absolute  necessity  to  good  work.  These 
directions  are  given  to  aid  in  understanding  the  methods,  but  no  one 
should  attempt  the  Breed  method  without  working  at  least  a  day  with 
someone  versed  in  the  technique. 

The  picture  one  sees  when  looking  into  the  microscope  is  hard 
to  describe.  There  may  be  rods  and  cocci,  as  well  as  objects  appearing 
similar  to  these.  One  has  to  become  accustomed  to  what  is  supposed 
to  be  seen.     The  cells  must  be  counted  in  each  of  thirty  fields,  the 


5  There  are  many  brands  of  stain  upon  the  market  today,  and  some  of  these 
are  better  than  others.  To  protect  the  users  of  such  products  from  spurious  and 
cheap  articles  a  commission  headed  by  Dr.  H.  J.  Conn  examines  every  batch  of 
stain  produced  by  manufacturers.  If  the  stain  is  found  to  pass  the  rigid  inspec- 
tion of  the  committee  a  label  bearing  the  signature  of  Dr.  Conn  is  attached  to 
the  bottle.     Do  not  use  stains  not  bearing  this  label. 


24 


UNIVERSITY    OF    CALIFORNIA EXPERIMENT    STATION 


average  taken,  and  this  average  multiplied  by  300,000;  or  the  total 
for  thirty  fields  may  be  counted  and  this  total  multiplied  by  10,000. 
The  results  in  either  case  are  the  same.  Although  the  regulation 
number  of  fields  to  be  counted  is  30,  there  are  frequently  occasions 
when  averaging  but  5  fields  will  give  all  the  information  necessary. 
Practice  is  necessary  to  successfully  use  the  Breed  method. 


E—  Eyepiece 


Pinion  Henri  — 


[)   S     Stage 


SS  — Substage 


Inclination  J 


Mechanical  Features  of  Microscope 

Fig.  9. — The  parts  of  a  microscope. 

The  method  will  not  permit  too  close  separation  of  grades  of  milk. 
One  could  not,  in  other  words,  distinguish  between  a  milk  with  18,000 
bacteria  per  cc.  and  one  with  22,000.  But  for  grading  milk  into  larger 
groups  nothing  is  simpler,  nor  more  rapid.  For  each  locality  the 
ranges  of  a  passable  milk  would  change  slightly,  and  these  limits 
should  be  established  for  each  locality. 

In  the  beginning  of  this  discussion  on  the  Breed  method  it  was 
considered  that  a  parity  should  exist  between  it  and  the  established 


ClEC.  310]         BACTERIOLOGICAL   LABORATORY   FOR  DAIRY   PLANTS  25 

plate  method  upon  which  our  laws  and  regulations  have  been  based. 
Dr.  Breed  has  had  a  number  of  workers  study  the  method  to  obtain 
just  this  information.6  As  a  result  of  this  work,  it  is  now  thought 
that  the  direct  count  is,  as  a  rule,  four  times  greater  than  the  plate 
count.  This  being  accepted,  it  is  necessary  to  divide  by  four  the  count 
obtained  by  the  direct  method.  This  will  give  a  value  close  to  that 
of  the  plate  count. 

THE    FROST    LITTLE-PLATE    METHOD 

The  Frost  method  of  enumeration  of  bacteria  in  milk  is  a  com- 
bination of  the  plate  and  the  direct  methods.  Milk  and  agar  are 
mixed  in  equal  portions  and  1/20  of  a  cc.  is  spread  over  an  area  of 
4  sq.  cm.  This  slide  is  placed  in  the  incubator  until  the  colonies 
develop.  It  usually  takes  about  8  hours.  The  microscopic  colonies 
are  then  counted  after  the  film  is  dried  and  stained  as  in  the  method 
used  in  the  direct  count.  No  new  principle  is  put  to  use,  and,  since 
the  method  is  not  much  in  vogue  except  in  special  cases  where  large 
city  plants  hold  pasteurized  milk  before  delivery,  it  will  not  be  de- 
scribed in  detail. 

THE    REDUCTASE    TEST 

Many  dyes  when  added  to  milk  in  dilute  solutions  will  be  decolor- 
ized. Litmus  and  methylene  blue  will  do  this.  There  seems  to  be  a 
feeling  that  there  is  an  indirect  relation  of  the  rate  of  the  decoloriza- 
tion  and  the  number  of  bacteria;  the  fewer  the  bacteria  the  longer 
the  time  necessary  for  decolorization.  This  is  only  generally  true, 
since  not  all  bacteria  possess  this  property  of  decolorization.  It  is 
apparent  that  a  milk  might  have  a  large  number  of  bacteria  present 
and  still  not  be  decolorized.  However,  the  method  is  one  that  has 
found  considerable  support  on  the  continent  of  Europe  and  even  in 
this  country  the  idea  is  growing.  The  argument  usually  put  forward 
is  that  we  have  a  medium  in  which  the  bacteria  can  grow  best  (the 
milk  itself)  instead  of  a  highly  artificial  one  such  as  the  usual  nutrient 
agar.  Be  that  as  it  may,  the  classes  into  which  milk  is  grouped  has 
no  great  significance  is  California,  where  the  milk  is  of  a  better 
quality  generally  than  in  many  parts  of  the  country.  The  following 
classes  are  recognized  in  Europe : 

Class  1. — Good  milk,  not  decolorized  in  five  and  a  half  hours, 
containing,  as  a  rule,  less  than  one-half  million  bacteria  per  cubic 
centimeter. 


6  Robertson,  A.  H.     1921.     See  "Selected  list  for  further  reading.' ' 


26  UNIVERSITY    OF    CALIFORNIA EXPERIMENT    STATION 

Class  2. — Milk  of  fair  average  quality,  decolorized  in  less  than  five 
and  a  half  hours  but  not  less  than  two  hours,  containing  as  a  rule, 
one-half  to  four  million  bacteria  per  cubic  centimeter. 

Class  3. — Bad  milk,  decolorized  in  less  than  two  hours,  but  not 
less  than  twenty  minutes,  containing,  as  a  rule,  four  to  twenty  million 
bacteria  per  cubic  centimeter. 

Class  4. — Very  bad  milk,  decolorized  in  twenty  minutes  or  less, 
containing,  as  a  rule,  over  twenty  million  bacteria  per  cubic  centi- 
meter. 

However,  we  still  have  use  for  the  method  under  certain  conditions 
and  the  method  is  described. 

Methylene-Blue  Solution. — A  stock  solution  of  methylene  blue  is 
made  by  dissolving  1.1  grams  of  the  dry  powder  in  500  cc.  of  distilled 
water.  One  cc.  of  this  stock  solution  is  diluted  with  39  cc.  of  distilled 
water,  giving  a  concentration  of  1  part  of  dye  to  20,000  parts  of 
water.  One  cc.  of  this  added  to  10  cc.  of  milk  makes  a  final  dilution 
of  the  dye  in  the  milk  of  1  to  200,000.    The  color  is  a  robin's  egg  blue. 

Ten  cc.  of  milk  are  placed  in  the  test  tubes  and  1  cc.  of  the  methy- 
lene-blue  solution  added.  Sterile  tubes  are  not  a  necessity  but  they 
must  be  clean.  The  contents  are  thoroughly  mixed  and  placed  in  a 
water  bath  at  37.5°  C.  The  tubes  are  observed  at  intervals,  and  notes 
are  made  on  the  time  of  decolorization.  An  exact  agreement  between 
the  time  taken  to  reduce  the  dye  and  the  plate  count  is  not  to  be 
expected. 

There  are  other  methods  of  evaluating  the  bacteria  content  of  a 
sample  of  milk.  These  will  not  be  discussed,  because  they  are  little 
used.  Those  that  have  been  explained  are  the  ones  in  most  general 
use.  Each  has  advantages;  each  has  faults.  The  plate  method  is 
slow  but  accurate  if  carefully  done.  The  direct  count  is  rapid  but 
suffers  by  reason  of  the  factor  of  300,000  which  has  to  be  applied. 
The  little-plate  method  (Frost's)  has  the  inherent  disadvantages  of 
both  the  above.  The  reductase  test  has  a  splendid  future  by  virtue 
of  certain  research  that  is  being  done  in  some  of  the  laboratories  of 
this  country. 


ClRC.   310]         BACTERIOLOGICAL   LABORATORY    FOR   DAIRY   PLANTS  27 


SELECTED    LIST    FOR    FURTHER    READING 

Plate  Method 

EOBERTSON,   A.   H. 

1921.     The  relation  between  bacterial  counts  from  milk  as  obtained  by  micro- 
scopic and  plate  methods.   New  York  State  (Geneva)  Agr.  Exp.  Sta., 
Tech.  Bui.  86:3-21. 
Ayres,  S.  H.,  and  C.  S.  Mudge. 

1920.  Milk  powder  agar  for  the  determination   of  bacteria  in  milk.     Jour. 

Bact.,  5:  565-588. 
Brew,  J.  D.,  and  W.  D.  Dotterrer. 

1917.  The  number  of  bacteria  in  milk.     New  York  State  (Geneva)  Agr.  Exp. 

Sta.  Bui.  439:479-522. 
Brown,  J.  Howard. 

1921.  Hydrogen  ions,  titration,  and  the  buffer  index  of  bacteriological  media. 

Jour.  Bact.  6:555-567. 
Barnett,  G.  D.,  and  H.  S.  Chapman. 

1918.  Colorimetric    determination    of    reaction    of    bacteriologic   medium    and 

other  fluids.     Jour.  Amer.  Med.  Assoc,  70:1062-1063. 
Breed,  E.  S.,  and  W.  D.  Dotterrer. 

1916.     The  number  of  colonies  allowable  on   satisfactory  agar  plates.     New 
York  State   (Geneva)    Agr.  Exp.  Sta.,  Tech.  Bui.  53:3-11. 

Direct  Count 
Breed,  E.  S.,  and  J.  D.  Brew. 

1916.  Counting    bacteria    by    means    of    the    microscope.      New    York    State 

(Geneva)  Agr.  Exp.  Sta.,  Tech.  Bui.  49:3-31. 

1917.  The  control  of  bacteria  in  market  milk  by  direct  microscopic  examina- 

tion.    New  York  State   (Geneva)   Agr.  Exp.  Sta.,  Bui.  433:717-746. 
Whiting,  William  S. 

1923.     The  relation  between  the  clumps  of  bacteria  found  in  market  milk  and 

the  flora  of  dairy  utensils.     New  York  State    (Geneva)    Agr.   Exp. 

Sta.,  Tech.  Bui.  98:3-36. 
Breed,  E.  S. 

1926.  The  microscopic   appearance  of   market   milk  and   cream.     New   York 

State   (Geneva)  Agr.  Exp.  Sta.,  Tech.  Bui.  120:3-7. 
Newman,  E.  W. 

1927.  One  solution  technique  for  direct  microscopic  counting  of  bacteria  in 

milk.     Proc.  Soc.  Exp.  Biol,  and  Med.,  24:323-325. 

Reductase  Method 
Hastings,  E.  G. 

1919.  The  comparative  value  of  quantitative  and  qualitative  bacteriological 

methods  as  applied  to  milk  with  especial  consideration  of  the  methy- 
lene blue  reduction  test.     Jour.  Dairy  Sci.,  2:293-311. 


28  UNIVERSITY    OF    CALIFORNIA EXPERIMENT    STATION 

Hastings,  E.  G.,  A.  Davenport,  and  W.  H.  Wright. 

1922.  The  influence  of  certain  factors  on  the  methylene  blue  reduction  test 
for  determining  the  number  of  bacteria  in  milk.  Jour.  Dairy  Sci., 
5:438-454. 

Little-plate  Method 
Frost,  W.  D. 

1916.  Comparison  of  a  rapid  method  of  counting  bacteria  in  milk  with  the 

standard  plate  method.     Jour.  Infect.  Diseases,  19:273-287. 

1917.  Counting  the  living  bacteria  in  milk:    a  practical  test.     Jour.   Bact., 

2:567-583. 


STATION  PUBLICATIONS  AVAILABLE  FOR  FREE   DISTRIBUTION 


No. 

253.  Irrigation  and  Soil  Conditions  in  the 
Sierra    Nevada    Foothills,    California. 

262.  Citrus   Diseases   of   Florida    and    Cuba 

Compared   with    those    of   California. 

263.  Size  Grades  for  Ripe  Olives. 

268.   Growing  and  Grafting  Olive  Seedlings. 

273.  Preliminary  Report  on  Kearney  Vine- 
yard Experimental  Drain,  Fresno 
County,    California. 

276.  The  Pomegranate. 

277.  Sudan   Grass. 

278.  Grain    Sorghums. 

279.  Irrigation   of  Rice  in   California. 
283.  The  Olive  Insects  of  California. 
294.   Bean   Culture  in   California. 

304.  A  Study  of  the  Effects  of  Freezes  on 

Citrus   in    California. 
310.   Plum    Pollination. 

312.  Mariout  Barley. 

313.  Pruning      Young      Deciduous      Fruit 

Trees. 
319.  Caprifigs    and   Caprification. 

324.  Storage  of   Perishable  Fruit  at  Freez- 

ing Temperatures. 

325.  Rice     Irrigation     Measurements     and 

Experiments    in    Sacramento   Valley, 

1914-1919. 
328.   Prune   Growing   in   California. 
331.   Phylloxera-Resistant    Stocks. 
335.   Cocoanut   Meal    as    a    Feed   for   Dairy 

Cows   and   Other  Livestock. 

339.  The    Relative    Cost    of    Making    Logs 

from   Small   and  Large  Timber. 

340.  Control     of     the     Pocket     Gopher     in 

California. 

343.  Cheese    Pests    and    Their    Control. 

344.  Cold   Storage   as   an   Aid  to   the   Mar- 

keting of  Plums. 

346.  Almond    Pollination. 

347.  The  Control  of  Red  Spiders  in  Decid- 

uous Orchards. 

348.  Pruning  Young  Olive  Trees. 

349.  A    Study    of    Sidedraft    and    Tractor 

Hitches. 

350.  Agriculture      in      Cut-over      Redwood 

Lands. 

353.  Bovine    Infectious    Abortion. 

354.  Results  of  Rice  Experiments  in   1922. 

357.  A     Self-mixing    Dusting    Machine    for 

Applying      Dry      Insecticides      and 
Fungicides. 

358.  Black    Measles,    Water    Berries,     and 

Related  Vine  Troubles. 

361.  Preliminary   Yield   Tables   for    Second 

Growth   Redwood. 

362.  Dust  and  the  Tractor  Engine. 

363.  The  Pruning  of  Citrus  Trees  in  Cali- 

fornia. 

364.  Fungicidal    Dusts    for   the    Control    of 

Bunt. 

365.  Avocado  Culture  in  California. 

366.  Turkish  Tobacco  Culture,   Curing  and 

Marketing. 

367.  Methods  of  Harvesting  and  Irrigation 

in   Relation  of  Mouldy  Walnuts. 

368.  Bacterial  Decomposition  of  Olives  dur- 

ing  Pickling. 

369.  Comparison     of     Woods     for     Butter 

Boxes. 

370.  Browning  of  Yellow  Newtown  Apples. 

371.  The    Relative    Cost   of   Yarding    Small 

and   Large  Timber. 

373.  Pear   Pollination. 

374.  A  Survey  of  Orchard  Practices  in  the 

Citrus    Industry  of    Southern     Cali- 
formia. 

375.  Results   of   Rice   Experiments   at   Cor- 

tena,    1923. 

376.  Sun-Drying  and  Dehydration  of  Wal- 

nuts. 

377.  The  Cold   Storage  of   Pears. 
379.  Walnut   Culture   in   California. 


BULLETINS 
No. 


380. 

382. 

385. 
386. 

387. 
388. 

389. 
390. 

391. 

392. 
393. 
394. 

S95. 
396. 

397. 

398. 
399. 


400. 
401. 

402. 
404. 
405. 
406. 
407. 


408. 
409. 


410. 


412. 


414. 


415. 
416. 


417. 
418. 


419. 
420. 


421. 
422. 


423. 
424. 


425. 
426. 


427. 
428. 


429. 


Growth  of  Eucalyptus  in  California 
Plantations. 

Pumping  for  Drainage  in  the  San 
Joaquin    Valley,    California. 

Pollination    of    the    Sweet    Cherry. 

Pruning  Bearing  Deciduous  Fruit 
Trees. 

Fig  Smut. 

The  Principles  and  Practice  of  Sun- 
drying  Fruit. 

Berseem  or  Egyptian   Clover. 

Harvesting  and  Packing  Grapes  in 
California. 

Machines  for  Coating  Seed  Wheat  with 
Copper   Carbonate   Dust. 

Fruit    Juice    Concentrates. 

Crop  Sequences  at  Davis. 

Cereal  Hay  Production  in  California. 
Feeding  Trials  with  Cereal  Hay. 

Bark   Diseases  of  Citrus  Trees. 

The  Mat  Bean  (Phaseolus  aconitifo- 
lius). 

Manufacture  of  Roquefort  Type  Cheese 
from   Goat's   Milk. 

Orchard  Heating  in  California. 

The  Blackberry  Mite,  the  Cause  of 
Redberry  Disease  of  the  Himalaya 
Blackberry,    and    its   Control. 

The  Utilization  of  Surplus  Plums. 

Cost  of  Work  Horses  on  California 
Farms. 

The  Codling  Moth  in  Walnuts. 

The  Dehydration  of  Prunes. 

Citrus  Culture  in  Central  California. 

Stationary  Spray  Plants  in  California. 

Yield,  Stand  and  Volume  Tables  for 
White  Fir  in  the  California  Pine 
Region. 

Alternaria  Rot  of  Lemons. 

The  Digestibility  of  Certain  Fruit  By- 
products as  Determined  for  Rumi- 
nants. 

Factors  Affecting  the  Quality  of  Fresh 
Asparagus  after  it  is  Harvested. 

Paradichlorobenzene  as  a  Soil  Fumi- 
gant. 

A  Study  of  the  Relative  Values  of  Cer- 
tain Root  Crops  and  Salmon  Oil  as 
Sources  of  Vitamin  A  for  Poultry. 

Planting  and  Thinning  Distances  for 
Deciduous  Fruit  Trees. 

The  Tractor  on   California   Farms. 

Culture  of  the  Oriental  Persimmon 
in    California. 

Poultry  Feeding :  Principles  and 
Practice. 

A  Study  of  Various  Rations  for 
Finishing  Range  Calves  as  Baby 
Beeves. 

Economic  Aspects  of  the  Cantaloupe 
Industry. 

Rice  and  Rice  By-products  as  Feeds 
for   Fattening    Swine. 

Beef   Cattle   Feeding   Trials,    1921-24. 

Cost  of  Producing  Almonds  in  Cali- 
fornia ;   a  Progress  Report. 

Apricots  (Series  on  California  Crops 
and  Prices). 

The  Relation  of  Rate  of  Maturity  to 
Egg  Production. 

Apple   Growing   in   California. 

Apple  Pollination  Studies  in  Cali- 
fornia. 

The  Value  of  Orange  Pulp  for  Milk 
Production. 

The  Relation  of  Maturity  of  Cali- 
fornia Plums  to  Shipping  and 
Dessert    Quality. 

Economic  Status  of  the  Grape  Industry. 


CIRCULARS 

No.  No. 

87.  Alfalfa.  259. 

117.  The    Selection    and    Cost    of    a    Small  261. 

Pumping  Plant.  262. 

127.  House    Fumigation.  263. 

129.  The  Control  of  Citrus  Insects.  264. 
136.  Melilotus    indica    as    a    Green-Manure 

Crop  for  California.  265. 

144.  Oidium    or    Powdery    Mildew    of    the  266. 

Vine. 

157.  Control  of  the  Pear  Scab.  267. 
164.   Small  Fruit  Culture  in  California. 

166.  The  County  Farm  Bureau.  269. 

170.  Fertilizing     California     Soils    for    the  270. 

1918   Crop.  272. 
173.  The    Construction    of   the    Wood-Hoop 

Silo.  273. 

178.  The  Packing  of  Apples  in   California.  276. 

179.  Factors    of    Importance   in    Producing  277. 

Milk  of  Low  Bacterial  Count. 

202.  County    Organizations   for   Rural   Fire  278. 

Control. 

203.  Peat   as   a   Manure   Substitute.  279. 
209.  The  Function  of  the  Farm  Bureau. 

212.   Salvaging    Rain-Damaged    Prunes.  281. 
215.  Feeding  Dairy  Cows  in  California. 
217.  Methods   for  Marketing  Vegetables   in 

California.  282. 

230.  Testing  Milk,   Cream,   and   Skim   Milk 

for  Butterfat.  283. 

231.  The    Home   Vineyard.  284. 

232.  Harvesting    and    Handling    California  285. 

Cherries    for    Eastern    Shipment.  286. 

234.  Winter  Injury  to  Young  Walnut  Trees  287. 

during  1921-22.  288. 

238.  The  Apricot  in  California.  289. 

239.  Harvesting     and     Handling     Apricots  290. 

and  Plums  for  Eastern  Shipment.  291. 

240.  Harvesting    and    Handling    Pears    for 

Eastern   Shipment.  292. 

241.  Harvesting  and  Handling  Peaches  for  293. 

Eastern    Shipment.  294. 

243.  Marmalade  Juice  and  Jelly  Juice  from  295. 

Citrus  Fruits. 

244    Central  Wire  Bracing  for  Fruit  Trees.  296. 
245.  Vine  Pruning  Systems. 

248.  Some   Common    Errors    in   Vine  Prun-  298. 

ing  and  Their  Remedies. 

249.  Replacing    Missing    Vines.  300. 

250.  Measurement   of   Irrigation   Water  on  301. 

the  Farm.  302. 

252.  Supports   for  Vines.  303. 

253.  Vinevard  Plans. 

254.  The  Use  of  Artificial  Light  to  Increase  304. 

Winter    Egg    Production.  305. 

255.  Leguminous   Plants  as  Organic  Fertil-  306. 

izer    in    California    Agriculture. 

256.  The   Control    of   Wild    Morning    Glory.  307. 

257.  The  Small-Seeded  Horse  Bean.  308. 

258.  Thinning  Deciduous   Fruits.  309. 


Pear  By-products. 

Sewing  Grain  Sacks. 

Cabbage  Growing  in  California. 

Tomato  Production  in  California. 

Preliminary      Essentials      to      Bovine 

Tuberculosis  Control. 
Plant   Disease  and   Pest  Control. 
Analyzing     the     Citrus     Orchard     by 

Means   of   Simple  Tree   Records. 
The  Tendency  of  Tractors  to   Rise  in 

Front";    Causes   and   Remedies. 
An  Orchard  Brush  Burner. 
A  Farm  Septic  Tank. 
California  Farm  Tenancy  and  Methods 

of  Leasing. 
Saving  the  Gophered  Citrus  Tree. 
Home  Canning. 
Head,    Cane,    and   Cordon   Pruning  of 

Vines. 
Olive  Pickling  in  Mediterranean  Coun- 
tries. 
The  Preparation  and  Refining  of  Olive 

Oil   in    Southern    Europe. 
The  Results  of  a  Survey  to  Determine 

the  Cost  of  Producing  Beef  in  Cali- 
fornia. 
Prevention  of  Insect  Attack  on  Stored 

Grain. 
Fertilizing  Citrus  Trees  in  California. 
The  Almond   in   California. 
Sweet  Potato  Production  in  California. 
Milk  Houses  for  California  Dairies. 
Potato   Production   in   California. 
Phylloxera   Resistant  Vineyards. 
Oak  Fungus  in  Orchard  Trees. 
The  Tangier  Pea. 
Blackhead   and   Other  Causes  of  Loss 

of  Turkeys  in  California. 
Alkali   Soils. 

The    Basis   of   Grape    Standardization. 
Propagation   of  Deciduous   Fruits. 
The   Growing   and    Handling  of   Head 

Lettuce   in   California. 
Control     of     the     California     Ground 

Squirrel. 
The    Possibilities    and    Limitations    of 

Cooperative   Marketing. 
Coccidiosis  of  Chickens. 
Buckeye  Poisoning  of  the  Honey  Bee. 
The   Sugar   Beet   in   California. 
A  Promising  Remedy  for  Black  Measles 

of  the   Vine. 
Drainage  on   the  Farm. 
Liming  the  Soil. 
A  General  Purpose  Soil  Auger  and  its 

Use  on  the  Farm. 
American   Foulbrood   and  its   Control. 
Cantaloupe  Production   in  California. 
Fruit  Tree  and  Orchard  Judging. 


The  publications  listed  above  may  be  had  by  addressing 

College  of  Agriculture, 

University  of  California, 

Berkeley,  California. 

10m-ll,'27 


